From ancient times, dogs have been favorably treated as pets by humans. In modern Europe and America, they are called "Companion species" and are now becoming a members of human society. On the other hand, dogs have hitherto been used as an experimental animal in various fields including medicine, pharmacy, animal husbandry, veterinary, psychology etc. They are also used as a SPF dog in recent years in tests for the determination of the effect and safety of drugs, and hence, the usefulness thereof for humans is becoming greater and greater. In any case, it is earnestly desired to establish a method for more certain diagnosis, treatment and prevention of canine diseases, especially canine infectious diseases in order to maintain healthy conditions thereof.
There are many canine viral diseases, and among them, those caused by canine distemper virus, canine parvovirus, infectious canine hepatitis, etc. are acute diseases having a very high mortality rate. Although vaccines for the prevention of these diseases have been developed, only symptomatic therapy such as antibiotics and sulfonamides which prevents secondary bacterial infections has been available for treating those dogs infected and attacked with these diseases, and hence, the conventional methods for treating these diseases are still insufficient.
Hitherto, a hyperimmune serum and an immunoglobulin derived from serum have been utilized for treatment of these diseases and confirmed to be effective. However, with the popularity of the idea for the kind treatment of animals, canine serum materials have become hard to obtain, and hence, this treatment can not be used nowadays. Therefore, development of a monoclonal antibody capable of neutralizing the infected viruses in place of the conventional hyperimmune serum will greatly contribute to the treatment of these viral diseases.
As mentioned above, a monoclonal antibody having a neutralizing activity against viruses can be used as an alternative to the hyperimmune serum. Hitherto, basic techniques of preparing monoclonal antibodies have been established mainly for a mouse monoclonal antibody.
Monoclonal antibodies produced by cells such as hybridomas can advantageously be obtained in a large amount and semipermanently and resolve the problem of material insufficiency. However, the monoclonal antibody in this case should be a canine monoclonal antibody instead of the conventional mouse monoclonal antibody in order to eliminate side effects such as anaphylatic shock, serum disease, etc. caused by the use in dogs of the mouse monoclonal antibody which acts as a heteroprotein to dogs.
Methods for preparing such canine monoclonal antibody as a drug for treating the canine viral diseases include:
(1) a method using a dog-dog hybridoma;
(2) a method using a canine lymphocyte transformed with some viral or chemical agent;
(3) a method using a dog-mouse heterohybridoma;
(4) a method using a dog-(dog-mouse) hybridoma derived from a dog-mouse heterohybridoma; and
(5) a method by gene recombination techniques of a mouse (V)-dog (C) chimeric monoclonal antibody wherein a variable (V) region which binds to an antigen is derived from a mouse monoclonal antibody having neutralizing activity against viruses and a constant (C) region which is responsible for antigenicity, immunogenicity and physiological activity is derived from a canine monoclonal antibody.
However, none of the above methods have hitherto been reported to be effectively used.
In the method (1), a fusion efficiency is quite low and no appropriate myeloma strain is available. In case of the method (2), there are no appropriate virus corresponding to EB virus in the case of humans and no appropriate chemical agents. The methods (3) and (4) will have much difficulty (for example, a stability problem etc.) in obtaining the desired canine monoclonal antibody with high efficiency in view of the case of preparation of a human monoclonal antibody. Therefore, it is expected that the method (5) using the chimeric monoclonal antibody is the most realizable method among these five methods.
The chimeric monoclonal antibody is prepared by incorporating a plasmid vector containing a mouse (V)-dog (C) chimeric antibody gene into an animal host cell (e. g. mouse myeloma cell), expressing said gene in the host cell and collecting the monoclonal antibody from a supernatant of the culture, wherein said mouse (V)-dog (C) chimeric antibody gene is such that a V (variable) gene is cloned from a mouse-mouse hybridoma capable of producing a mouse monoclonal antibody as a source of a gene coding for a V region, a C (constant) gene is cloned from a canine cell such as a canine antibody-producing cell capable of producing a canine monoclonal antibody as a source of a gene coding for a C region and said V gene and said C gene are linked to each other. Several reports are found as to human chimeric antibodies (Japanese Patent First Publication Nos. 155132/1985 and 47500/1986).
As mentioned above, a gene coding for an amino acid sequence in a variable (V) region of an antibody molecule capable of binding to a desired antigen and a gene coding for an amino acid sequence in a constant (C) region of a canine immunoglobulin are require for preparing the canine chimeric antibody. The gene coding for the variable (V) region of the chimeric antibody is derived from a cell capable of producing a mouse monoclonal antibody having a neutralizing activity against the above mentioned various canine viruses and said cell can be prepared rather easily by the conventional mouse-mouse hybridoma producing procedures. However, the gene coding for the constant (C) region of the chimeric antibody, i. e. the gene coding for the constant (C) region of the canine immunoglobulin is still unknown in its structure and has never been cloned. Therefore, in order to prepare the canine chimeric antibody, it is inevitably required to find the gene coding for the amino acid sequence of the constant (C) region of the canine immunoglobulin.
In addition, although there is much difficulty for obtaining the monoclonal antibody showing a desired specificity in case of the methods (1) to (4), materials (cell strains) effective for preparing the chimeric antibody can effectively be provided in the case of the method (5) since any cell which produces the canine immunoglobulin regardless of its specificity can preferably be employed as materials for providing a gene coding for the C region of the canine immunoglobulin for preparing the chimeric antibody.